Methods and pharmaceutical compositions for inducing immune tolerance by mucosal vaccination with fc-coupled antigens

ABSTRACT

The present invention relates to methods and pharmaceutical compositions of inducing immune tolerance by mucosal vaccination with Fc-coupled antigens. In particular, the present invention relates to a method for inducing tolerance to one antigen of interest in a subject in need thereof, comprising the mucosal administration to the subject of a therapeutically effective amount of a recombinant chimeric construct comprising a FcRn targeting moiety and an antigen-containing moiety.

FIELD OF THE INVENTION

The present invention relates to methods and pharmaceutical compositionsof inducing immune tolerance by mucosal vaccination with Fc-coupledantigens.

BACKGROUND OF THE INVENTION

Autoimmune diseases represent a major health concern. For example, type1 diabetes (T1D) is an autoimmune disease mediated by autoreactive Tcells that recognize β-cell antigens (Ags), leading to destruction ofpancreatic islets. A major problem in T1D management is its latediagnosis. This typically takes place after a variable period ofsubclinical, silent autoimmunity, once a significant proportion of βcells have already been destroyed. The resulting insulin deficiencyleads to hyperglycemia and clinical onset. Hence, T1D prevention andtreatment should target the underlying autoimmune mechanisms rather thanits metabolic consequences, as done today with insulin replacementtherapies. However, immunotherapies aimed at blunting β-cellautoimmunity need to have an excellent safety profile, since T1D mostlyaffects children and young adults and is not life-threatening in theshort term.

β-cell Ag-specific therapies are therefore attractive in light of theirselectivity and safety, compared to treatments broadly targeting theT-cell subsets involved in disease¹. Such therapies are administered inthe form of vaccines comprising the β-cell Ags whose immune recognitionmediates islet destruction. These vaccines are formulated to achieveimmune outcomes that are opposite to those pursued with classicalvaccination, i.e. to neutralize rather than to stimulate the T-cellresponses against the administered Ags—instating a condition known asimmune tolerance. Clinical trials have however been deceiving²⁻⁴.Several attempts have focused on tolerogenic vaccination with β-cell Agsderived from insulin and its precursor preproinsulin (PPI), since thisis the initiating Ag in the non-obese diabetic (NOD)^(5,6) mouse andlikely also in humans². A recent trial employing intranasal insulinadministration to halt autoimmune β-cell destruction in new-onset T1Dpatients with slowly evolving disease did not result in significantβ-cell preservation, despite evidence that insulin-specific immunetolerance was successfully induced⁷. These results suggest that we mayneed to intervene earlier, before significant β-cell loss and beforeautoimmune progression. While PPI recognition initiates the autoimmunecascade, subsequent β-cell destruction releases additional Ags that arefurther recognized (a phenomenon known as Ag spreading), thus makingtolerance restoration vis-à-vis of the sole PPI insufficient.

The same problem is encountered in prevention trials using insulinvaccination, where the safety issue is even more critical for treatingat-risk subjects who are not yet diabetic. Despite absence of clinicaldisease, selection of at-risk subjects is based on positivity formultiple auto-antibodies (auto-Abs), which witness an autoimmunereaction that already involves several Ags^(3,8,9). Recent studiesfurther suggest that β-cell autoimmunity initiates very early, as themedian age at auto-Ab seroconversion was only 9-18 months in largeprospective cohorts of genetically at-risk children^(10,11). InsulinAg-specific prevention strategies should therefore be implemented at amuch earlier stage, in at-risk children (i.e. first-degree relatives ofT1D patients) carrying a high HLA-associated genetic risk of disease butwith no signs of active autoimmunity (i.e. auto-Ab-)^(12,13).

The perinatal period offers such opportunities not only in terms oftiming, but also because it is characterized by immune responses tointroduced Ags that are biased towards tolerogenic outcomes. Indeed, Agintroduction during fetal and neonatal life results in Ag-specificimmune tolerance persisting during adulthood¹⁴⁻¹⁶. A key role in thisprocess is played by central tolerance, a process taking place in thethymus in which developing T cells are challenged with ectopicallyexpressed self-tissue Ags such as PPI. Their recognition leads toelimination of autoreactive pathogenic T effector cells (Teffs) and topositive selection of T regulatory cells (Tregs)¹⁷. This process is veryactive during the perinatal period and leads to the definition ofimmunological self that later imprints peripheral immune responses. The‘immune self-image’ presented in the thymus is however incomplete,because the self Ag repertoire is only partially expressed¹⁷. Indeed,defective central tolerance is the first checkpoint in T1D progression.Some autoreactive Teffs escape thymic selection and can later beactivated in the periphery and perpetrate islet destruction. Supportingthis notion, the NOD mouse model of T1D develops accelerated diabeteswhen PPI expression is abolished in the thymus (Ins2−/−NODmice)^(18,19). Second, human INS polymorphic variants predispose to T1Dby decreasing INS expression in the thymus²⁰.

However, this knowledge has not translated into therapeutic strategiesaimed at boosting central tolerance ab initio. All tolerogenicvaccination approaches explored to date using the subcutaneous,intranasal or oral route are targeted on peripheral tolerance and aim atblunting the pathogenic potential of autoreactive Teffs and/or atenhancing Treg activity¹. If we could instead introduce self Ags such asPPI in the thymus during the perinatal period, we could boost the T-cellselection process and intervene on the very first step in autoimmuneprogression.

The present invention relates to the use of Ags fused with the Fcportion of an IgG to induce immune tolerance by mucosal vaccination.Fc-fusion proteins currently represent 20% of all Ab-based medicineswith FDA approval and are actively investigated in a variety of settingsbecause addition of an Fc moiety increases the half-life of proteintherapeutics. Binding of the Fc domain to the neonatal Fc receptor(FcRn) expressed in endothelial cells leads to their transientintracellular sequestration and slow release in the circulation. TheFcRn is a heterodimer composed of a major histocompatibility complex(MHC) Class-I-like heavy chain and β2-microglobulin²¹. The interactionbetween IgG and FcRn requires an acidic pH (<6.5) and is inefficient ata physiological pH (7.4)²¹. It occurs in a 1:2 stoichiometry, with oneIgG binding to two FcRn molecules via the FcRn heavy chains and theCH2-CH3 portion of the Fc domain of IgG²².

Besides endothelial cells, the FcRn is expressed in several othertissues and cells²³, including the placental syncytiotrophoblast or yolksac of mammals, the liver, intestinal, bronchial, renal (proximalconvoluted tubule), genital, ocular and choroid plexus epithelia, renalpodocytes; the skin (hair follicles, sebaceous glands, epidermalkeratinocytes and melanocytes); hematopoietic cells such as dendriticcells, monocytes and macrophages (including macrophages in the laminapropria of the small intestine)²⁴. In the respiratory tract, the FcRn ispredominantly found in the bronchial epithelium of upper and centralairways. In the human digestive tract, FcRn is expressed in epithelialcells of the stomach, the small intestine (duodenum, jejunum and ileum)and the colon²⁵⁻²⁷. There is an increasing proximal-distal gradient ofmucosal FcRn mRNA and protein expression in the intestinal tract, withexpression being the lowest in the duodenum-jejunum and highest in theproximal colon. This expression gradient correlates with the efficiencyof in vitro monoclonal Ab (mAb) transcytosis for these differentintestinal regions, with systemic entry occurring via the lymphatics²⁷.The same expression gradient is found in cynomolgus monkeys, in whichserum mAb levels were greater after ileum-proximal colon infusion thanafter administration into the duodenum-jejunum²⁷. Taken together, theFcRn expression and mAb uptake patterns suggest that the ileum-proximalcolon is the region where most of the mAb is transcytosed through theFcRn.

In more recent years, the application of Fc-coupled agents has thereforebeen extended to strategies aimed at delivering bioactive moleculesusing less invasive administration route, namely the gastrointestinal orpulmonary route. Typically, IgG are endocytosed on the apical membraneof epithelial cells and bind to FcRn at the acidic pH present inendosomes. The vesicle then fuses again with the basolateral membrane,where the extracellular neutral pH promotes the dissociation of IgG fromFcRn. Importantly, FcRn-mediated transport of IgG is bidirectional (i.e.both from the apical and basolateral membrane of epithelial cells)²⁸ andoccurs rapidly, within 1 h after IgG addition in in vitro Transwellexperiments²⁶. Examples of Fc-coupled agents explored for oral deliveryinclude Fc-coupled follicle-stimulating hormone (FSH)²⁹, IgG mAbs²⁷ andFc-coated nanoparticles containing bioactive molecules such asinsulin³⁰. Overall, the systemic bioavailability achieved by oraladministration of Fc-coupled agents is relatively limited. Examples ofFc-coupled agents explored for pulmonary delivery include Fc-couplederythropoietin³¹⁻³³ and Fc-coupled FSH²⁹.

Hence, Fc-coupled agents have been used to increase the half-life ofsystemically administered therapeutic proteins and to facilitate theirsystemic delivery through the intestinal or bronchial route. We herepropose to use the same strategy to induce immune tolerance. To thisend, Ags can be modified by fusing them to the Fc portion of an IgG1,thus allowing the resulting Ag-Fc proteins to interact with the FcRn andcross mucosal barriers. This is the same pathway that physiologicallydelivers maternal IgG to foetuses (through the placenta) and to newborns(through the gut, during lactation)²¹, thus providing passive IgGprotection during the intrauterine period and the first 6 months oflife, when IgG production is not yet operational. This concept was firstvalidated using the transplacental route of transfer and indicated thatFc-fused Ags intravenously administered to pregnant mice reach the fetalthymus in an FcRn-dependent manner and promote the generation ofAg-specific Tregs, leading to Ag-specific tolerance³⁴. When applied toT1D mouse models, PPI-Fc transplacental transfer protects the offspringfrom diabetes development later in life^(35,36). A single 100 μg PPI-Fcdose intravenously administered to pregnant PPI T-cellreceptor-transgenic G9C8 NOD mice (in which all T cells recognize aPPI_(B15-23) epitope³⁷) is transplacentally transferred and protects theoffspring from diabetes, without inducing metabolic or other adverseeffects. This transfer is Fc- and FcRn-dependent. Diabetes protection isassociated with peripheral PPI-reactive CD8⁺ Teffs displaying impairedcytotoxicity and with increased thymic-derived neuropilin-1(NRP1/CD304)⁺ CD4⁺ Tregs secreting the regulatory cytokine transforminggrowth factor (TGF)-β. PPI-Fc reaches the thymus carried by migratory(CD8^(lo)CD11b⁺SIRPα⁺) dendritic cells (DCs) and diabetes protection islost when migration is inhibited by early administration ofanti-vascular cell adhesion molecule (VCAM)-1 Abs. Importantly, diabetesprotection is also active in polyclonal NOD mice³⁵. Although successful,this strategy remains invasive for autoimmune diseases like T1D that wecannot predict with certainty early enough, a fortiori prenatally. Suchstrategies applied to pregnant women may be considered to carry a riskfor the fetus and the mother unacceptable in front of diseases that arein most cases not life-threatening in the short term and that may or maynot develop.

SUMMARY OF THE INVENTION

The present invention relates to methods and pharmaceutical compositionsfor inducing immune tolerance by mucosal vaccination with Fc-coupledAgs. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

Now the inventors have evidence that antigen-Fc orally administereddirectly to newborn mice induces profound T-cell modificationssuggestive of tolerance induction. The inventors therefore propose toexplore its suitability for preventing or treating diseases andconditions such as autoimmune diseases, allergic diseases and immuneresponses against antigens that are exogenously administered.

Thus, one object of the present invention relates to a method forinducing tolerance to one antigen of interest in a subject in needthereof comprising the mucosal administration to the subject of atherapeutically effective amount of a recombinant chimeric constructcomprising a FcRn targeting moiety and an antigen-containing moiety.

As used herein, the term “antigen” has its general meaning in the artand generally refers to a substance or fragment thereof that isrecognized and selectively bound by an antibody or by a T cell antigenreceptor, resulting in induction of an immune response. Antigensaccording to the invention are typically, although not exclusively,peptides and proteins. Antigens may be natural or synthetic andgenerally induce an immune response that is specific for that antigen.Other non-protein antigens recognized by specialized T-cell subsets suchas natural killer T cells³⁸⁻⁴⁰ and mucosal-associated invariant Tcells⁴¹⁻⁴³ may also be considered.

In some embodiments, the antigen is an auto-antigen. As used herein, theterm “auto-antigen” means any self-antigen arising from the own bodytissues which is mistakenly recognized by the immune system as beingforeign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

In some embodiments, the antigen is an allergen. As used herein, theterm “allergen” generally refers to an antigen or antigenic portion of amolecule, usually a protein, which elicits an allergic response uponexposure to a subject. Typically the subject is allergic to the allergenas indicated, for instance, by the wheal and flare test or any methodknown in the art. A molecule is said to be an allergen even if only asmall subset of subjects exhibit an allergic immune response uponexposure to the molecule.

In some embodiments, the antigens are molecules that are exogenouslyadministered for therapeutic or other purposes and may trigger anunwanted immune response. While frequently neutralising the biologicalactivity that said molecules are meant to induce, such immune responsesmay have additional deleterious effects unrelated to the purpose forwhich the molecules were originally administered. Examples of this kindinclude immune reactions against therapeutic clotting factor VIII inhaemophilia A or factor IX in haemophilia B, against different enzymesin congenital enzymopathies and, more in general, during any kind ofreplacement therapies in the context of genetic deficiencies.Allo-immunization responses against antigens expressed by tissues orhematopoietic and/or blood cells transplanted into an individual areequally considered.

The term “tolerance,” as used herein, refers to a failure to respond, ora reduced response, to an antigen (including auto-antigens, allergensand endogenously administered molecules). This may mean that aproductive (immunogenic) response is not induced upon endogenous orexogenous exposure to said antigen. This response may be replaced, inpart or completely, by a tolerogenic response, i.e. an active processthat further limits immunogenic responses. Examples of tolerogenicresponses include, but are not limited to, generation of T regulatorycells, elimination of effector (conventional) T cells by apoptosis ortheir neutralization by anergy, and skewing of T cells and other immunecells towards phenotypes favouring a state of tolerance, e.g. productionof regulatory cytokines such as interleukin-10 and TGF-β and of otheranti-inflammatory mediators and downregulated expression ofco-stimulatory molecules. These immunological concepts are well known tothe skilled in the art.

In some embodiments, the subject is human, but treatment of otheranimals is also encompassed. In some embodiments, the subject is anadult. In some embodiments, the subject is a pregnant woman. In someembodiments, the subject is a child. In some embodiments, the subject isa child that is less than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 year(s) old. In some embodiments, the subject is anewborn. In some embodiments, the subject is a neonate. As used herein,a “neonate” is a newborn that is less than about 28-day-old. In someembodiments, the subject is a pregnant woman that may give birth to achild at risk of developing a disease.

In some embodiments, the subject is predisposed or believed to bepredisposed to developing, or has already developed or is developing, atleast one symptom of a disease or condition caused by inappropriate orunwanted immune system activity against an antigen. The subject may beidentified or diagnosed as having done so or as likely to do so based ona variety of factors, for example, family history and/or genetic testingof e.g. the mother and/or father, siblings, other relatives(grandparents, aunts, uncles, cousins, etc.), presence of other diseasebiomarkers such as (auto)antibodies directed against different(self-)antigens. Generally, the subject is known to have a geneticpredisposition to development of an autoimmune disease, an allergy orother unwanted immune response. By “is known to have a geneticpredisposition”, we mean that one or both parents or siblings may havethe disease or condition, and/or are known to be carriers of gene(s)that is/are associated with the disease or condition, so that thestatistical probability of the subject having or developing the diseaseis at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90%, or is 100%. Thedetermination may be based on observation of the health of the parents,siblings or of the subject, or on genetic testing of the same andidentification of a gene or genes in a form known to be associated withor to cause the disease or condition, e.g. to have a particular sequencesuch as an allele, mutation, insertion, deletion, etc. The risk ofdisease may or may not also be confirmed by genotypying subject's cellsand/or by assessing them by suitable biomarkers, including non-geneticbiomarkers such as, by way of example, antibodies and other immunephenotypes or epigenetic modifications. Those of skill in the art willalso recognize that such genetic traits may not be “all or nothing”, inthat gene dosage may apply. Nevertheless, if a subject is deemed to beat risk, and if the life of a subject can be lengthened or improved bythe practice of the present methods, then the subject is a viablecandidate for treatment.

In some embodiments, the subject is predisposed or believed to bepredisposed to developing, or has already developed or is developing anautoimmune disease. As used herein, the term “autoimmune disease” refersto the presence of an autoimmune response (an immune response directedagainst an auto- or self-antigen) in a subject. Autoimmune diseasesinclude diseases caused by a breakdown of self-tolerance such that theadaptive immune system, in concert with cells of the innate immunesystem, responds to self-antigens and mediates cell and tissue damage.In some embodiments, autoimmune diseases are characterized as being aresult of, at least in part, a humoral and/or cellular immune response.Examples of autoimmune disease include, without limitation, acutedisseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagicleukoencephalitis, Addison's disease, agammaglobulinemia, alopeciaareata, amyloidosis, ankylosing spondylitis, anti-GBM/Anti-TBMnephritis, antiphospholipid syndrome (APS), autoimmune angioedema,autoimmune aplastic anemia, autoimmune dysautonomia, autoimmunehepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency,autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmunepancreatitis, autoimmune retinopathy, autoimmune thrombocytopenicpurpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonaland neuronal neuropathies, Behcet's disease, bullous pemphigoid,autoimmune cardiomyopathy, Castleman disease, celiac disease, Chagasdisease, chronic fatigue syndrome, chronic inflammatory demyelinatingpolyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO),Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosalpemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease,congenital heart block, coxsackie myocarditis, CREST disease, essentialmixed cryoglobulinemia, demyelinating neuropathies, dermatitisherpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica),discoid lupus, Dressler's syndrome, endometriosis, eosinophilicfasciitis, erythema nodosum, experimental allergic encephalomyelitis,Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis(temporal arteritis), glomerulonephritis, Goodpasture's syndrome,granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barresyndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolyticanemia, Henoch-Schonlein purpura, herpes gestationis,hypogammaglobulinemia, hypergammaglobulinemia, idiopathicthrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosingdisease, immunoregulatory lipoproteins, inclusion body myositis,inflammatory bowel disease, insulin-dependent diabetes (type 1),interstitial cystitis, juvenile arthritis, Kawasaki syndrome,Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus,lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD),lupus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis,mixed connective tissue disease (MCTD), monoclonal gammopathy ofundetermined significance (MGUS), Mooren's ulcer, Mucha-Habermanndisease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy,neuromyelitis optica (Devic's), autoimmune neutropenia, ocularcicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS(Pediatric Autoimmune Neuropsychiatric Disorders Associated withStreptococcus), paraneoplastic cerebellar degeneration, paroxysmalnocturnal hemoglobinuria (PNH), Parry Romberg syndrome,Parsonnage-Turner syndrome, pars planitis (peripheral uveitis),pemphigus, peripheral neuropathy, perivenous encephalomyelitis,pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, &III autoimmune polyglandular syndromes, polymyalgia rheumatica,polymyositis, postmyocardial infarction syndrome, postpericardiotomysyndrome, progesterone dermatitis, primary biliary cirrhosis, primarysclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathicpulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia,Raynaud' s phenomenon, reflex sympathetic dystrophy, Reiter's syndrome,relapsing polychondritis, restless legs syndrome, retroperitonealfibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidtsyndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicularautoimmunity, stiff person syndrome, subacute bacterial endocarditis(SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis,temporal arteritis/Giant cell arteritis, thrombocytopenic purpura (TTP),Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis,undifferentiated connective tissue disease (UCTD), uveitis, vasculitis,vesiculobullous dermatosis, vitiligo, Waldenstrom's macroglobulinemia(WM), and Wegener's granulomatosis [Granulomatosis with Polyangiitis(GPA)]. In some embodiments, the autoimmune disease is selected from thegroup consisting of rheumatoid arthritis, type 1 diabetes, systemiclupus erythematosus (lupus or SLE), myasthenia gravis, multiplesclerosis, scleroderma, Addison's Disease, bullous pemphigoid, pemphigusvulgaris, Guillain-Barré syndrome, Sjogren syndrome, dermatomyositis,thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonalgammopathy of undetermined significance (MGUS), Waldenstrom'smacroglobulinemia (WM), chronic inflammatory demyelinatingpolyradiculoneuropathy (CIDP), Hashimoto's Encephalopathy (HE),Hashimoto's Thyroiditis, Graves' Disease, Wegener's Granulomatosis[Granulomatosis with Polyangiitis (GPA)]. In some embodiments, theautoimmune disease is type 1 diabetes.

In some embodiments, the subject is predisposed or believed to bepredisposed to developing, or has already developed or is developing anallergy. As used herein, the term “allergy” generally refers to aninappropriate immune response characterized by inflammation andincludes, without limitation, food allergies, respiratory allergies andother allergies causing or with the potential to cause a systemicresponse such as, by way of example, Quincke's oedema and anaphylaxis.The term encompasses allergy, allergic disease, hypersensitiveassociated disease or respiratory disease associated with airwayinflammation, such as asthma or allergic rhinitis. In some embodiments,the method of the present invention is effective in preventing, treatingor alleviating one or more symptoms related to anaphylaxis, drughypersensitivity, skin allergy, eczema, allergic rhinitis, urticaria,atopic dermatitis, dry eye disease, allergic contact allergy, foodhypersensitivity, allergic conjunctivitis, insect venom allergy,bronchial asthma, allergic asthma, intrinsic asthma, occupationalasthma, atopic asthma, acute respiratory distress syndrome (ARDS) andchronic obstructive pulmonary disease (COPD). Hypersensitivityassociated diseases or disorders that may be treated by the method ofthe present invention include, but are not limited to, anaphylaxis, drugreactions, skin allergy, eczema, allergic rhinitis, urticaria, atopicdermatitis, dry eye disease [or otherwise referred to asKeratoconjunctivitis sicca (KCS), also called keratitis sicca,xerophthalmia], contact allergy, food allergy, allergic conjunctivitis,insect venom allergy and respiratory diseases associated with airwayinflammation, for example, IgE mediated asthma and non-IgE mediatedasthma. The respiratory diseases associated with airway inflammation mayinclude, but are not limited to, rhinitis, allergic rhinitis, bronchialasthma, allergic (extrinsic) asthma, non-allergic (intrinsic) asthma,occupational asthma, atopic asthma, exercise induced asthma,cough-induced asthma, acute respiratory distress syndrome (ARDS) andchronic obstructive pulmonary disease (COPD).

In some embodiments, the subject is predisposed or believed to bepredisposed to developing, or has already developed or is developing animmune reaction against molecules that are exogenously administered fortherapeutic or other purposes and may trigger an unwanted immuneresponse. Non-limiting examples of this kind include immune reactionsagainst replacement therapeutics in the context of genetic deficiencies,which include, but are not limited to, haemophilia A, haemophilia B,congenital deficiency of other clotting factors such as factor II,prothrombin and fibrinogen, primary immunodeficiencies (e.g. severecombined immunodeficiency, X-linked agammaglobulinemia, IgA deficiency),primary hormone deficiencies such as growth hormone deficiency andleptin deficiency, congenital enzymopathies and metabolic disorders suchas disorders of carbohydrate metabolism (e.g. sucrose-isomaltasedeficiency, glycogen storage diseases), disorders of amino acidmetabolism (e.g. phenylketonuria, maple syrup urine disease, glutaricacidemia type 1), urea cycle disorders (e.g. carbamoyl phosphatesynthetase I deficiency), disorders of organic acid metabolism (e.g.alcaptonuria, 2-hydroxyglutaric acidurias), disorders of fatty acidoxidation and mitochondrial metabolism (e.g. medium-chain acyl-coenzymeA dehydrogenase deficiency), disorders of porphyrin metabolism (e.g.porphyrias), disorders of purine or pyrimidine metabolism (e.g.Lesch-Nyhan syndrome), disorders of steroid metabolism (e.g. lipoidcongenital adrenal hyperplasia, congenital adrenal hyperplasia),disorders of mitochondrial function (e.g. Kearns-Sayre syndrome),disorders of peroxisomal function (e.g. Zellweger syndrome), lysosomalstorage disorders (e.g. Gaucher's disease, Niemann Pick disease). In thecase of genetic deficiencies, the proposed method may not only allow toreinstate immune tolerance against the replacement therapeutics that areused to treat the disease, but also reinstate the biological activityfor which said therapeutics are administered. Other therapeutics forwhich said method may be suitable to limit undesired immune responsesinclude other biological agents such as, by way of example, cytokines,monoclonal antibodies, receptor antagonists, soluble receptors, hormonesor hormone analogues, coagulation factors, enzymes, bacterial or viralproteins. For example, haemophilic children can be treatedprophylactically with periodic coagulation factor (e.g. factor VIII)replacement therapy, which decreases the chance of a fatal bleed due toinjury. In addition to the expense and inconvenience of such treatment,repeated administration results in inhibitor antibody formation in somepatients against the coagulation factor. If the antibodies in thesepatients are low titer antibodies, patients are treated with largerdoses of blood coagulation factors. If the antibodies are high titerantibodies, treatment regimens for these patients become much morecomplex and expensive. In some embodiments, the therapeutic protein isproduced in the subject following gene therapy suitable e.g. for thetreatment of congenital deficiencies. Gene therapy typically involvesthe genetic manipulation of genes responsible for disease. One possibleapproach for patients, like those with haemophilia deficient for asingle functional protein, is the transmission of genetic materialencoding the protein of therapeutic interest. However, the repeatedadministration of gene therapy vectors, such as viral vectors, may alsotrigger unwanted immune responses against the therapeutic proteinintroduced in the vector and/or against other components of the vector.Thus, the method of the present invention can be suitable for overcomingthe body's immune response to gene therapy vectors such as viralvectors. Viral vectors are indeed the most likely to induce an immuneresponse, especially those, like adenovirus and adeno-associated virus(AAV), which express immunogenic epitopes within the organism. Variousviral vectors are used for gene therapy, including, but not limited to,retroviruses for X-linked severe combined immunodeficiency (X-SCID);adenoviruses for various cancers; adeno-associated viruses (AAVs) totreat muscle and eye diseases; lentivirus, herpes simplex virus andother suitable means known in the art.

In some embodiments, the subject is predisposed or believed to bepredisposed to developing, or has already developed or is developing animmune reaction against a grafted tissue or grafted hematopoietic cellsor grafted blood cells. Typically the subject may have been transplantedwith a graft selected from the group consisting of heart, kidney, lung,liver, pancreas, pancreatic islets, brain tissue, stomach, largeintestine, small intestine, cornea, skin, trachea, bone, bone marrow,muscle, or bladder. The method of the present invention is alsoparticularly suitable for preventing or suppressing an immune responseassociated with rejection of a donor tissue, cell, graft, or organtransplant by a recipient subject. Graft-related diseases or disordersinclude graft versus host disease (GVHD), such as associated with bonemarrow transplantation, and immune disorders resulting from orassociated with rejection of organ, tissue, or cell grafttransplantation (e.g., tissue or cell allografts or xenografts),including e.g., grafts of skin, muscle, neurons, islets, organs,parenchymal cells of the liver, etc. Thus the method of the invention isuseful for preventing Host-Versus-Graft-Disease (HVGD) andGraft-Versus-Host-Disease (GVHD). The chimeric construct may beadministered to the subject before, during and/or after transplantation(e.g., at least one day before transplantation, at least one day aftertransplantation, and/or during the transplantation procedure itself). Insome embodiments, the chimeric construct may be administered to thesubject on a periodic basis before and/or after transplantation.

In some embodiments, the method of the present invention is particularlysuitable for the treatment of autoimmune diseases, allergic diseases andcongenital deficiencies.

As used herein, the term “treatment” or “treat” refer to bothprophylactic or preventive treatment as well as curative or diseasemodifying treatment, including treatment of patient at risk ofcontracting the disease or suspected to have contracted the disease aswell as patients who are ill or have been diagnosed as suffering from adisease or medical condition, and includes suppression of clinicalrelapse. The treatment may be administered to a subject having a medicaldisorder or who ultimately may acquire the disorder, in order toprevent, cure, delay the onset of, reduce the severity of, or ameliorateone or more symptoms of a disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment. By “therapeutic regimen” is meant the pattern oftreatment of an illness, e.g., the pattern of dosing used duringtherapy. A therapeutic regimen may include an induction regimen and amaintenance regimen. The phrase “induction regimen” or “inductionperiod” refers to a therapeutic regimen (or the portion of a therapeuticregimen) that is used for the initial treatment of a disease. Thegeneral goal of an induction regimen is to provide a high level of drugto a patient during the initial period of a treatment regimen. Aninduction regimen may employ (in part or in whole) a “loading regimen”,which may include administering a greater dose of the drug than aphysician would employ during a maintenance regimen, administering adrug more frequently than a physician would administer during amaintenance regimen, or both. The phrase “maintenance regimen” or“maintenance period” refers to a therapeutic regimen (or the portion ofa therapeutic regimen) that is used for the maintenance of a patientduring treatment of an illness, e.g., to keep the patient in remissionfor long periods of time (months or years). A maintenance regimen mayemploy continuous therapy (e.g., administering a drug at regularintervals, e.g., daily, weekly, monthly, yearly, etc.) or intermittenttherapy (e.g., interrupted treatment, intermittent treatment, treatmentat relapse, or treatment upon achievement of a particular predeterminedcriteria [e.g., disease manifestation, etc.]).

According to the present invention, the chimeric construct comprises anantigen-containing moiety. This moiety comprises an antigenic moleculeor a portion of an antigenic molecule for which is it desired togenerate immune tolerance, and may comprise a plurality of antigens orportions of antigens. Preferably, the antigen is a protein, apolypeptide or a peptide. In some embodiments, the antigen-containingmoiety comprises one or more known epitopes of interest, e.g. regions orresidues of the antigen which are known to elicit an immune response.Alternatively, putative epitopes and antigenic regions may be selectedbased on a likelihood of antigenicity due to accessibility, surfaceexposure, charge, amino acid sequence etc. e.g. using predictionsoftware programs well known in the art. Typically, theantigen-containing moiety comprises contiguous sequences of the primarysequence of an antigen. Alternatively, sufficient residues of theantigen may be present so that secondary and tertiary structure is atleast partially preserved, and antigenic regions are present that arenot necessarily contiguous in primary sequence but are adjacent afterfolding of the molecule or generated by fusion of non-adjacent antigensequences, as described for different ‘hybrid’ epitopes. The antigenicmoiety of the construct may contain an epitope or multiple epitopes fromone or from more than one antigen of interest. Multiple epitopes may becontinuous in the construct sequence or separated by appropriatelinkers. The multiple epitopes may be the same, e.g. multiple copies ofthe same epitope may be present; or the epitopes may be different, e.g.single or multiple copies of two or more different epitopes may bepresent. The epitopes may be “different” from (may differ from) eachother either by virtue of originating from different antigenicmolecules, or by originating from different parts of the same antigenicmolecule, or both, e.g. the same region of an antigen from severaldifferent variants may be used. Combinations of the above may also bepresent. Post-translationally modified epitopes or epitopes generated byalternative splicing may equally be included. The amino acid sequence ofthe antigen-containing moiety may have 100% identity to that of aprotein, polypeptide or peptide that is a natural antigen.Alternatively, the antigenic moiety may comprise a portion (e.g. atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95% or more) of the residues of a native sequence to whichimmune tolerance is desired. Further, the amino acid sequence in theantigenic moiety may be the same primary sequence as that of a nativeprotein antigen, or may be a variant thereof with at least about 50, 55,60, 65, 70, 75, 80, 85, 90, 95% or more identity or homology to thenative protein sequence, or to the portion of the native sequence onwhich it is based.

In some embodiments, the antigen-containing moiety derives frompreproinsulin (PPI), glutamic acid decarboxylase (GAD),insulinoma-associated protein 2 (IA-2), islet-specificglucose-6-phosphatase catalytic-subunit-related protein (IGRP), zinctransporter 8 (ZnT8), pre-pro-islet amyloid polypeptide (ppIAPP), 78 kDaglucose-regulated protein [GRP78 and its precursor; also known as heatshock 70 kDa protein 5 (HSPA5)], dystrophia myotonica kinase (DMK) andchromogranin A for T1D; myeloperoxydase and proteinase 3 forgranulomatosis with polyangiitis; myelin oligodendrocyte glycoprotein(MOG), myelin basic protein (MBP) and proteolipid protein (PLP) inmultiple sclerosis; various synovial antigens such as vimentin, nuclearribonucleoprotein-A2 (RA33), fibrinogen, alpha-enolase for rheumatoidarthritis; tissue transglutaminase and gliadins in celiac disease.Post-translationally modified epitopes, alternative splice isoforms andhybrid peptides derived from said antigens—alone or in combination withother antigens—are equally included. Canonical antigens or antigenisoforms that are not properly expressed in the thymus may beparticularly suitable to this end. Examples of antigen moieties arederived from the above said protein following processing byantigen-presenting cells—including dendritic cells—and presentation inthe context of different human leukocyte antigen (HLA) Class I or ClassII molecules. Therefore, said peptide antigens are different dependingnot only on their source antigen, but also on the HLA molecules by whichthey are presented. For example, a list of T1D-associated peptideantigens for both mouse and human can be found in DiLorenzo et al.,Clin.Exp.Immunol. 148:1, 2007⁴⁴.

In some embodiments, the antigen-containing moiety derives from anallergen. Allergens include, but are not limited to. phospholipase A2(API ml) associated with severe reactions to bee, Derp-2, Der p 2, Der f, Der p 5 and Der p 7 associated with reaction against the house-dustmite Dermatophagoides pteronyssinus, the cockroach allergen Bla g 2 andthe major birch pollen allergen Bet v 1.

In some embodiments, the antigen-containing moiety derives from atherapeutic protein. The term “therapeutic proteins” as used hereinrefers to protein or peptide compounds of any aminoacid length that areadministered or are planned to be administered in vivo to human subjectsto achieve a therapeutic effect. Examples of such therapeutic proteinsare, but are not limited to, antibodies of different species (either intheir native form or partially/fully humanized), cytokines, receptorantagonists, soluble receptors, hormones or hormone analogues,coagulation factors, enzymes, bacterial or viral proteins. Example oftherapeutic applications wherein the therapeutic protein can be suitableinclude, without being limited to, cytokine- and antibody-based immunetherapies, hormone replacement therapies and replacement therapies forcoagulation factors (e.g., factor VIII in haemophilia A) or enzymaticdeficits (e.g., beta-glucuronidase in mucopolysaccharidosis VII). In allthese situations, mounting of immunogenic responses against theadministered protein is not desirable, as this would becounterproductive for achieving the desired therapeutic effect (e.g.,side effects such as cytokine release syndromes; orneutralization/degradation of the therapeutic protein).

As used herein, the term “neonatal Fc receptor” or “FcRn” has itsgeneral meaning in the art and refers to the neonatal Fc receptor whichis an Fc receptor. Unlike FcγRs which belong to the Immunoglobulinsuperfamily, human FcRns structurally resemble polypeptides of MajorHistocompatibility Complex (MHC) Class I⁴⁵. FcRn is typically expressedas a heterodimer consisting of a transmembrane α or heavy chain incomplex with a soluble β or light chain (β2 microglobulin). FcRn shares22-29% sequence identity with Class I MHC molecules has a non-functionalversion of the MHC peptide binding groove⁴⁶. Like MHC, the α chain ofFcRn consists of three extracellular domains (α1, α2, α3) and a shortcytoplasmic tail that anchors the protein to the cell surface. The α1and α2 domains interact with FcR binding sites in the Fc region ofantibodies⁴⁷.

In some embodiments, the FcRn targeting moiety is typically a protein orpolypeptide that is capable of binding to and mediating uptake of theentire construct by the FcRn receptor. Generally, the FcRn targetingmoiety is an Fc of an IgG antibody, preferably of an IgG1 or IgG4antibody, even more preferably of an IgG1 antibody, or a portion of theFc that is sufficient to permit binding and uptake of the construct. Asused herein, the term “Fc region” includes amino acid sequences derivedfrom the constant region of an antibody heavy chain. The Fc region isthe portion of a heavy chain constant region of an antibody beginning atthe N-terminal of the hinge region at the papain cleavage site, at aboutposition 216 according to the EU index and including the hinge, CH2, andCH3 domains. Exemplary Fc regions or portions thereof that may be usedin the practice of the invention are well known in the art.

In some embodiments, the recombinant chimeric construct of the presentinvention is a fusion protein that comprises an amino acid sequenceconsisting of a portion of an Fc region (e.g., the portion of the Fcregion that confers binding to FcRn) and an amino acid sequence of anon-immunoglobulin polypeptide that comprises the antigenic portion ofthe antigen.

As used herein, the term “fusion protein” refers to a chimericpolypeptide which comprises a first amino acid sequence linked to asecond amino acid sequence with which it is not naturally linked innature. As used herein, the terms “linked,” “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two ormore elements or components, by whatever means including chemicalconjugation or recombinant means. An “in-frame” or “operably linked”fusion refers to the joining of two or more open reading frames (ORFs)to form a continuous longer ORF, in a manner that maintains the correctreading frame of the original ORFs. Thus, the resulting recombinantfusion protein is a single protein containing two or more segments thatcorrespond to polypeptides encoded by the original ORFs (which segmentsare not normally so joined in nature.) Although the reading frame isthus made continuous throughout the fused segments, the segments may bephysically or spatially separated by, for example, an in-frame linkersequence. Various detectable labels may be additionally included tofacilitate production of the fusion construct or its detection onceadministered in vivo. In addition, various other functionalities may beincluded in the constructs. Examples of such functionalities include,but are not limited to, domains of the antigenic protein that arerequired to exert one or several desired biological activities (e.g.binding to receptor(s) or avoidance of such binding) or that aremodified so to increase such biological activities. The fusion proteinof the present invention may be produced by any method well known in theart, for example, by chemical synthesis, or by creating and translatinga polynucleotide in which the peptide regions are encoded in the desiredrelationship.

In some embodiments, the Fc region of the fusion protein includessubstantially the entire Fc region of an antibody, beginning in thehinge region just upstream of the papain cleavage site which defines IgGFc chemically (about residue 216 EU numbering, taking the first residueof heavy chain constant region to be 114) and ending at its C-terminus.The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity, secretion, or binding characteristics of themolecule. Methods for making fusion proteins are known in the art. Asused herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain, e.g. fromabout position 216-230 according to the EU number system. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains⁴⁸. As used herein, theterm “CH2 domain” includes the portion of a heavy chain molecule thatextends, e.g., from about EU positions 231-340. The CH2 domain is uniquein that it is not closely paired with another domain. Rather, twoN-linked branched carbohydrate chains are interposed between the two CH2domains of an intact native IgG molecule. As used herein, the term “CH3domain” includes the portion of a heavy chain molecule that extendsapproximately 110 residues from N-terminus of the CH2 domain, e.g., fromabout residue 341-446, EU numbering system). The CH3 domain typicallyforms the C-terminal portion of the antibody. In some immunoglobulins,however, additional domains may extend from CH3 domain to form theC-terminal portion of the molecule (e.g. the CH4 domain in the chain ofIgM and the E chain of IgE).

In some embodiments, the Fc region of the fusion protein does notinclude the hinge region but comprises the CH2 and CH3 domains that isfused to the amino acid sequence that comprises the antigenic portion ofthe antigen.

Further methods of reducing the size of the constructs may also beemployed, such as those described in U.S. patent applications2002/0155537, 2007/0014794, and 2010/0254986 (each to Carter et al.),and 2014/0294821 (Dumont et al.). For example, Fc-Fc andantigen-Fc/antigen-Fc dimer formation may be prevented.

In some embodiments, the Fc region may be mutated in order to increasethe binding affinity or specificity for the FcRn. Examples of suchmutations are summarized in recent reviews²³ and include, but are notlimited to, H435A⁴⁹, N434A⁵⁹ and M428L modifications⁵¹. FcRn-bindingpeptides that achieve similar or superior effects as compared to thenative Fc domain have also been described⁵² and may be used forgenerating said fusion proteins together with the antigen(s) ofinterest, further allowing to decrease the relative molecular weight ofthe added FcRn-binding moiety. In some embodiments, the Fc region may bemutated in order to limit enzymatic degradation, e.g. from pepsin.

In some embodiments, the FcRn-binding moieties are coupled to thesurface of nanoparticles, and the antigen(s) of interest are enclosed insuch nanoparticles. Examples of nanoparticles include, but are notlimited to, biodegradable and biocompatible poly(lacticacid)—bpoly(ethylene glycol) (PLA-PEG) block copolymers³⁰. TheFcRn-binding moieties can be coupled to the nanoparticle surface bysuitable methods, e.g. using ring-opening polymerization with a freeterminal maleimide group. This strategy may have the advantage ofprotecting said antigens from degradation. Procedures to producesuitable nanoparticles are known in the art, examples can be found in³⁰.

By a “therapeutically effective amount” of the chimeric construct of thepresent invention as above described is meant a sufficient amount ofsaid construct to reach a therapeutic effect. It will be understood,however, that the total daily usage of the compounds and compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular subject will depend upon avariety of factors including the disorder being treated and the severityof the disorder; activity of the specific compound employed; thespecific composition employed, the age, body weight, general health, sexand diet of the subject; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination orcoincidentally with the specific inhibitor employed; and like factorswell known in the medical arts. For example, it is well within the skillof the art to start doses of the compound at levels lower than thoserequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved. However, thedaily dosage of the products may be varied over a wide range from 0.01to 1,000 mg per adult per day. Typically, the compositions contain 0.01,0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500mg of the active ingredient for the symptomatic adjustment of the dosageto the subject to be treated. A medicament typically contains from about0.01 mg to about 500 mg of the active ingredient, preferably from 1 mgto about 100 mg of the active ingredient. An effective amount of thedrug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7mg/kg of body weight per day.

As used herein, the term “mucosal administration” includes any form ofadministration of the recombinant chimeric construct of the presentinvention through a mucosal surface that expresses neonatal Fcreceptors. The term “mucosal” refers to a tissue comprising a mucousmembrane, such as, but not limited to, nasal mucosa, pulmonary mucosa,oral mucosa (including sublingual, oral, buccal, enteral, intestinal,rectal or gastric) or vaginal mucosa. In particular, the termencompasses pulmonary administration and oral administration. In someembodiments, the recombinant chimeric construct of the present inventionis delivered via the oral cavity. In some embodiments, the recombinantchimeric construct is delivered via the respiratory tract, e.g. byintranasal delivery, inhalation and any method known in the art. Therecombinant chimeric construct of the present invention is typicallyadministered to the subject in the form of any pharmaceuticalcomposition that is compatible with the mucosal route of administration.For example, the recombinant chimeric construct of the present inventioncan be administered as a solution or suspension together with apharmaceutically acceptable carrier. Such a pharmaceutically acceptablecarrier can be, for example, water, phosphate buffered saline, normalsaline or other physiologically buffered saline, or other solvent orvehicle such as glycol, glycerol, and oil such as olive oil or aninjectable organic ester. A pharmaceutically acceptable carrier can alsocontain liposomes or micelles prepared by mixing the recombinantchimeric construct of the present invention with detergent and aglycoside, such as Quil A. In some embodiments, the recombinant chimericconstruct of the present invention is combined with the carrier in anyconvenient and practical manner, e.g., by solution, suspension,emulsification, admixture, encapsulation, absorption and the like. Suchprocedures are routine for those skilled in the art. In someembodiments, the recombinant chimeric construct of the present inventionis combined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can also be added in the mixing process in order toprotect the composition from loss of therapeutic activity, e.g.,denaturation in the stomach. Examples of stabilizers for use in thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc., proteolytic enzymeinhibitors, and the like. The composition for oral administration can befurther formulated into hard or soft shell gelatin capsules, tablets, orpills. More preferably, gelatin capsules, tablets, or pills areenterically coated. Enteric coatings prevent denaturation of thecomposition in the stomach or upper bowel where the pH is acidic. See,e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, thebasic pH therein dissolves the coating and permits the recombinantchimeric construct of the present invention to be released and absorbedby specialized cells, e.g., epithelial enterocytes.

In some embodiments, the chimeric construct may be further protected bydegradation by expressing it in biologically contained Lactococcuslactis or other suitable bacteria, alone or in combination withimmunomodulatory molecules which include, but are not limited to,interleukin-10 and transforming growth factor β. Such formulations maycarry the additional advantage of bypassing the need for synthesizingthe needed chimeric construct as a recombinant protein of suitableclinical grade and of achieving a steady, low-dose delivery ofantigen-Fc constructs that may be more effective at restoring long-termtolerance⁵³. This methods are known in the art and examples can be foundin⁵³⁻⁵⁵. Thus the method of the present invention encompasses the use ofa recombinant bacteria wherein in the genome of which has been inserteda nucleic acid encoding for the recombinant chimeric construct of thepresent invention. A recombinant bacteria according to the invention maybe selected from the group comprising Lactobacillus, Leuconostoc,Pediococcus, Lactococcus, Streptococcus, Escherichia, Streptococcus,Agrobacterium, Bacillus, Corynebacterium, Clostridium, Gluconobacter,Citrobacter, Enterobacter, Klebsiella, and Pseudomonas. In someembodiments, a recombinant bacteria according to the invention is aprobiotic lactic acid bacteria, in particular of the Lactococcus genus,and more particularly of the Lactococcus lactis species.

In some embodiments, the chimeric construct of the present invention isadministered via the airways, e.g. into the nasal cavity, trachea orlungs. Typically the chimeric construct of the present invention isdelivered by any device adapted to introduce a therapeutic compositioninto the upper and/or lower respiratory tract. In some embodiments, thedevices of the present invention may be metered-dose inhalers. Thedevices may be adapted to deliver the therapeutic compositions of theinvention in the form of a finely dispersed mist of liquid, foam orpowder. The device may use a piezoelectric effect or ultrasonicvibration to dislodge powder attached on a surface such as a tape inorder to generate mist suitable for inhalation. The devices may use anypropellant system known to those in the art including, but not limitedto, pumps, liquefied gas, compressed gas and the like. Devices typicallycomprise a container with one or more valves through which the flow ofthe therapeutic composition travels and an actuator for controlling theflow. In particular, the devices suitable for administering theconstructs of the invention include inhalers and nebulisers such asthose typically used to deliver steroids to asthmatics. In some cases,where the subject is for example a child, a spacer may be used tofacilitate effective administration from the inhaler. Various designs ofinhalers are available commercially and may be employed to deliver themedicaments of the invention. These include, without being bound totheory, the Accuhaler, Aerohaler, Aerolizer, Airmax, Akita Jet,Autohaler, Clickhaler, Diskhaler, Easi-breathe inhaler, Fisonair,Integra, Jet inhaler, Miat-haler, Novolizer inhaler, Pari Boy, Pulvinalinhaler, Rotahaler, Spacehaler, Spinhaler, Syncroner inhaler andTurbohaler devices.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1. Schematic of the delivery strategy. The physiological pathwaythat transfers maternal breastmilk IgG to the newborn is exploited. Thispathway is FcRn-dependent, and fusion of preproinsulin (PPI) with an IgGFc fragment grants it access to the bloodstream through the intestinalepithelium.

FIG. 2. Orally administered PPI-Fc remains confined to the gut in theabsence of FcRn. One-day-old C56BL/6 wild-type (FcRn wt) and FcRn−/−mice were force-fed with 50 μg of Alexa-labeled PPI-Fc and imaged after72 h. PPI-Fc fluorescence remains detectable in the gut of FcRn−/− butnot FcRn wt mice.

FIG. 3. Systemic and thymic PPI-Fc delivery upon oral administration.One-day-old PPI T-cell receptor (TCR)-transgenic G9C8 NOD newborn micewere force-fed with 50 μg of Alexa-labeled PPI-Fc or PPI and imagedafter 72 h. PPI-Fc but not PPI accumulation is detected at the wholebody level (top) and in the thymus (bottom). Results refer to arepresentative experiment out of two performed.

FIG. 4. Serum concentrations of PPI-Fc and PPI upon oral administration.ELISA quantification on serum samples collected at the indicated timepoints following force-feeding at day 1 as above. Data are depicted asmean+SEM.

FIG. 5A-B. Oral PPI-Fc administration induces immune tolerance. A.Percent spleen CD8⁺CD3⁺ and CD4⁺CD3⁺ T cells in 4-week-old G9C8 micetreated at 1 day of life as above. B. Percent spleen CD4⁺ T-cell subsetsin the same mice. Bars represent median and interquartile ranges. ***,p<0.001; **, p<0.01; *, p<0.05.

FIG. 6. Gating strategy for the analysis of antigen-presenting cells.Relevant gates are numbered as follows: 1, macrophages; 2, B cells; 3,plasmacytoid dendritic cells (DCs); 4, migratory CD103⁺CD11b⁻ cDCs; 5,resident CD103⁻CD11b⁻ SIRPα⁻ cDCs; 6, resident CD103⁻CD11b⁻SIRPα⁺ cDCs;7, migratory CD103⁺CD11b⁺ cDCs; 8, resident CD103⁻CD11b⁺ cDCs.Representative results from the spleen of a 2-week-old G9C8 mouse areshown.

FIG. 7. Gating strategy for the analysis of T-cell subsets. After gatingon viable CD3+ cells (not shown), CD8+ (panel A) and CD4+ T cells (panelF) were analysed for the expression of different markers. Relevant gatesare numbered as follows: 1, CD8⁺ T cells; 2, activated/memory CD8⁺ Tcells; 3, naïve CD8⁺ T cells; 4, CCR9+ activated CD8⁺ T cells; 5, α4β7⁺activated CD8⁺ T cells; 6, CCR9⁺ naïve CD8+ T cells; 7, α4β7⁺ naïve CD8⁺T cells; 8, CD4⁺ T cells; 9, activated/memory CD4⁺ T cells; 10, naïveCD4⁺ T cells; 11, CCR9⁺ activated CD4⁺ T cells; 12, α4β7⁺ activated CD4⁺T cells; 13, CCR9⁺ naïve CD4⁺ T cells; 14, α4β7⁺ naïve CD4⁺ T cells; 15,thymic-derived Tregs; 16, peripheral Tregs; 17, Th3 cells; 18, Tr1cells. Representative results from the spleen of a 2-week-old G9C8 mouseare shown.

FIG. 8. CCR9^(hi) CD8⁺ T cells in PPI-Fc vs. IgG1-treated G9C8 mice.A-B. Percent (A) and counts (B) of activated (CD44^(hi)CD62L⁻)CCR9^(hi)CD8⁺ T cells in 2-week-old G9C8 mice treated at 1 day of lifeas above. C-D. Percent (C) and counts (D) of naïve (CD44⁻CD62L⁺)CCR9^(hi)CD8⁺ T cells in the same mice. Bars represent medians. *,p<0.05.

FIG. 9. α4β7⁺ CD4⁺ T cells in PPI-Fc vs. IgG1-treated G9C8 mice. A-B.Percent (A) and counts (B) of activated (CD44^(hi)CD62L⁻) α4β7⁺CD4⁺ Tcells in 2-week-old G9C8 mice treated at 1 day of life as above. C-D.Percent (C) and counts (D) of naïve (CD44⁻CD62L⁺) α4β7⁺CD4⁺ T cells inthe same mice. Bars represent medians. *, p<0.05.

FIG. 10. Peripheral and thymic Tregs in PPI-Fc vs. IgG1-treated G9C8mice. A-B. Percent (A) and counts (B) of peripheral (NRP1⁻) Foxp3⁺CD4⁺Tregs in 2-week-old G9C8 mice treated at 1 day of life as above. C-D.Percent (C) and counts (D) of thymic (NRP1⁺) Foxp3⁺CD4⁺ Tregs in thesame mice. Bars represent medians. *, p<0.05.

FIG. 11. Orally delivered PPI-Fc protects from diabetes. Diabetesincidence in G9C8 mice force-fed at day 1 with 50 μg PPI-Fc (solidline), equimolar amounts of PPI (dashed line) or IgG1 (dotted line).Diabetes was subsequently induced by immunization with PPIB15-23 and CpGat 4 and 6 weeks of age. **P<0.01 by Mann Whitney U test.

EXAMPLE Material & Methods

Generation of Mouse PPI1-Fc and PPI2-Fc Fusion Proteins

Sequences encoding PPI1 and PPI2 were PCR-amplified from pancreatic andthymic cDNA, respectively, and inserted into pCR4-TOPO plasmids(Invitrogen)³⁵. Following digestion with the appropriate restrictionenzymes, PPI1/2 sequences were inserted at EcoRV/BglII sites by cohesiveend ligation into pFUSE-hIgG1-Fc2 expression vector (InvivoGen),downstream of an IL-2 signal peptide and upstream of the human Fcγ1sequence. PPI1-Fc and PPI2-Fc sequences were then re-amplified by PCRand ligated at XbaI/XhoI sites into the pFastBac1 expression vector(Invitrogen). These constructs were inserted into the Bac-to-BacBaculovirus Expression System (Invitrogen), expressed in Hi5 insectcells and protein products purified on Sepharose-coupled protein G (GEHealthcare). Protein identity was confirmed by reducing SDS-PAGE andWestern blot using rabbit anti-insulin polyclonal antibody (H-86, SantaCruz) and mouse anti-human Fc monoclonal Ab (Southern Biotech). PPI1 andPPI2 were purified from Hi5 insect cell pellets as previouslydescribed⁵⁶. PPI1-Fc (hereinafter referred to as PPI-Fc) was used in theexperiments depicted.

Mice

C56BL/6 wild-type and C56BL/6 FcRn−/− mice were obtained from theJanvier Labs and the Jackson Laboratory, respectively. G9C8 Cα^(−/−) NODmice are transgenic for a PPI_(B15-23) TCR and have been previouslydescribed and characterized^(35,37).

In Vivo PPI-Fc Imaging

PPI-Fc and PPI proteins were conjugated with Alexa Fluor (AF)680 usingSAIVI Rapid Antibody/Protein labeling kit (Invitrogen). One-day-oldnewborn mice were force-fed with 50 μg PPI-Fc or equimolar amounts ofPPI. Fluorescence was detected using the Fluobeam imaging system(Fluoptics) at a 690 nm excitation and >700 nm emission wavelengths,with 50-100 ms exposures.

ELISA Quantification of Serum PPI-Fc and PPI Concentrations

Following force-feeding as above, blood was collected at the indicatedtime points for ELISA quantification, with standard curves obtained bysequential dilutions of PPI-Fc and PPI proteins. Both PPI-Fc and PPIwere captured with plate-coated H-86 anti-insulin Ab (Santa Cruz).PPI-Fc was detected with a horseradish peroxidase-labeled goatanti-human Fc antibody (Southern Biotech). PPI was revealed with ananti-proinsulin monoclonal Ab (KL-1; kindly provided by Dr. L. Harrison,Walter and Eliza Hall Institute, Parkville, Australia).

Spleen T-Cell Phenotyping

The following monoclonal antobodies were used on splenocytes retrievedfrom treated mice: PE-labeled anti-Foxp3, APC-eFluor780-labeledanti-CD3ε (eBioscience); APC-labeled anti-neuropilin-1 (NRP1; R&D);Brilliant Violet (BV)605-labeled anti-CD4 and BV711-labeled anti-CD8a(BioLegend). Cells were additionally stained with Live/Dead Red(Invitrogen).

K^(d) Multimer Preparation

Monomers composed of the mouse MHC class I heavy chain H-2Kd, humanβ2-microglobulin and the PPI B15-23 peptide (LYLVCGERL) were synthesizedas described³⁵ and incubated with BV650-coupled streptavidin (mole:moleratio 4:1) for 1 h. D-biotin and bovine serum albumin were then added at25 μM and 0.5% concentrations, respectively. The obtained multimers(MMrs) were stored at 4° C. protected from light and used the same day.

Phenotyping of Subsets of Antigen presenting Cells and T Cells in theSpleen and Lymph Nodes.

All cells were washed and resuspended in PBS 1x prior to transfer into96-well V-bottom plated (200 μl/well) for FACS staining BD CompBeads (10μl/well, 1 drop in 500 μl PBS) were added to each well prior to stainingto normalize cell counts. Two antibody panels were used, as follows.

T-cell panel. Dasatinib (50 μM, 100 μl/well) was added to each cellpellet for 30 min at 37° C. After centrifugation, cells were resuspendedin 18 μl of MMr solution containing a 20% dilution of 50 μM dasatinibfor 20 min at room temperature, followed by incubation for 20 min at 4°C. with 18 μL of the following antibody premix: Live/DEAD Aqua(Invitrogen, 1/1,000), CD3-APC-eFluor780 (clone 145-2C11, eBioscience,1/100), CD4-BV711 (clone RM4-5, BD Biosciences, 1/200), CD8-AF700 (clone53-6.7, BD Biosciences, 1/150), CCR9-PE-Cy7 (clone CW-1.2, BioLegend,1/100); NRP1-APC (clone 3E12, BioLegend, 1/50), LPAM-1 (α4β7)-PE-CF594(clone ATK32, BD Biosciences, 1/100), latency-associated peptide(LAP)-BV421 (clone TW7-16B4, BioLegend, 1/200), CD62L-BV605 (cloneMEL-14, BD Biosciences, 1/200), CD44-BV786 (clone IM7, BD Biosciences,1/100). A second 10 μL antibody mix was then added for 15 min at 37° C.:CD49b-FITC (clone HMα2, BioLegend, 1/200), LAG3-PerCP-Cy5.5 (cloneC9B7W, BD Biosciences, 1/200). Cells were then washed in PBS, fixed andpermeabilised with the Foxp3 Fix/Perm Buffer Set (BioLegend), andincubated with 30 μl anti-Foxp3 antibody (Foxp3-PE, clone FJK-16s,eBioscience, 1/50 in FoxP3/Perm buffer). After a final wash, cells wereresuspended in 200 μl PBS 1x and kept at 4° C. prior to flow cytometryacquisition.

Antigen-presenting cell (APC) panel. An identical staining protocol wasapplied without fixation and permeabilisation, with an incubation for 20min at 4° C. with 30 μl of the following antibody mix: Live/DEAD Aqua(Invitrogen, 1/1,000), NK1.1-B V510 (clone PK136, BD Biosciences,1/100), CD3-BV510 (clone 145-2C11, BD Biosciences, 1/100), F4/80-BV711

(clone BM8, BioLegend, 1/50), CD8-AF700 (clone 53-6.7, BD Biosciences,1/150), B220-PE-Cy7 (clone RA3-6B2, BD Biosciences, 1/150),SIRPα-PerCP-eF1710 (clone P84, eBioscience, 1/50), PDCA-1-Pacific Blue(clone 927, BioLegend, 1/200), CD11b-BV650 (clone M1/70, BD Biosciences,1/150), CD11c-BV605 (clone N418, BioLegend, 1/50), CD103-BV786 (cloneM290, BD Biosciences, 1/100).

PPI-Fc Treatment, Diabetes Induction and Follow-Up

G9Cα−/−.NOD mice were force-fed on day 1 after birth with 50 μg PPI-Fcor control proteins, namely equimolar quantities of Fc-devoid PPI orHerceptin IgG1. For diabetes induction, 4-week-old mice were primed with50 μg PPI_(B15-23) peptide and 100 μg CpG 4 days after weaning, followedby a second identical immunization 15 days later. Diabetes developmentwas monitored by testing glycosuria and confirmed by glycaemia whenpositive. Diabetic mice were sacrificed by cervical dislocation.

Results

The Intestinal Transfer of Orally Administered PPI-Fc is FcRn- andFc-Dependent

A schematic of the strategy used is depicted in FIG. 1. We exploited theintestinal FcRn pathway that physiologically delivers breastmilk IgG tothe newborn. To this end, we fused the PPI1 or PPI2 protein with theN-terminus of the CH2-CH3 Fc domain from human IgG1 to obtain PPI1-Fcand PPI2-Fc fusion proteins. While Fc-devoid PPI1/2 is not able to crossthe intestinal epithelium, addition of the Fc moiety should favor thistransfer. We explored this strategy using a PPI1-Fc construct(hereinafter referred to as PPI-Fc).

We first performed ex vivo imaging on 1-day-old FcRn−/− and wild-typeC56BL/6 mice force-fed with fluorescently labeled PPI1-Fc and sacrificedafter 72 h (FIG. 2). The PPI-Fc fluorescence was still detectable in theintestines of FcRn−/− but not of wild-type mice, suggesting lack oftransfer in the absence of FcRn.

To verify the occurrence of such transfer, 1-day-old G9C8 newborn micewere force-fed with fluorescently labeled PPI-Fc or Fc-devoid PPI (FIG.3). After 72 h, systemic PPI-Fc accumulation was promptly visualized,which was not the case for PPI. PPI-Fc, but not PPI, was also visualizedin the thymus.

Serum PPI1-Fc concentrations (FIG. 4) were of ˜1 μg/ml at 24 h afteradministration (day 2) and remained relatively stable up to 72 h after(day 3; ˜0.75 μg/ml). PPI1 was not detected at any of these time points.

Collectively, these results show that oral administration of a single 50μg dose of PPI-Fc, but not of PPI, results in intestinal transfer,systemic antigen bioavailability and delivery to the thymus, which isFcRn- and Fc-dependent.

Oral PPI-Fc Vaccination induces Tolerogenic T-Cell Modifications.

We next assessed whether thymic PPI-Fc delivery induced T-cellmodifications compatible with immune tolerance (FIG. 5A). Splenocytesobtained at 4 weeks of age following treatment at day 1 of life as abovedisplayed significantly decreased numbers of CD8⁺ effector T cells inPPI-Fc-treated newborns compared with PBS-treated ones. No significantdifference was highlighted in the number of total CD4⁺ T cells betweenPPI-Fc- and PBS-treated mice. However, when analyzing CD4⁺ T cellsubsets, significant differences were detected (FIG. 5B). Compared withPBS-treated mice, PPI1-Fc-treated animals displayed decreased numbers ofCD4⁺ effector T cells (Foxp3⁻) and increased numbers of Foxp3⁺regulatory T cells (Tregs), both thymic-derived (NRP1/CD304⁻) andperipherally induced (NRP1/CD304⁻).

A second set of experiments was performed by comparing G9C8 mice forcefed with either PPI-Fc or IgG1, in order to differentiate the effectsinduced by the Fc moiety by those relying on the PPI antigenic portion.These mice were analyzed at 2 weeks of age, i.e. closer to the first dayof life at which they were orally vaccinated. Both APC and T-cellsubsets were analyzed in the spleen, mesenteric lymph nodes (MLNs) andpancreatic lymph nodes (PLNs).

The APC gating strategy is depicted in FIG. 6. After gating on forwardand side scatter and live cells (not shown), CD3⁻NK1.1⁻(lineage-negative) cells were selected to exclude T and NK cells,respectively (FIG. 6A). Three gates were then selected according to twomarkers: F4/80⁻B220⁻, F4/80⁻B220⁺ and F4/80⁻B220⁻ (FIG. 6B). F4/80⁺B220⁻cells were then plotted for CD11c and CD11b expression to identifyCD11c⁻CD11b⁺ macrophages (FIG. 6C, gate 1). Similarly, F4/80⁻B220⁺ cellswere plotted for CD11c and CD11b markers to gate CD11c⁻CD11b⁻ B cells(FIG. 6D, gate 2) and CD11b⁻CD11c⁺ cells, which reveal the plasmacytoiddendritic cell (pDC) population upon further gating for PDCA-1 marker(FIG. 6E, gate 3) and for the migration marker CD103. Among F4/80⁻B220⁻cells, two gates were selected according to CD11c and CD11b markers(FIG. 6F). Collectively, these CD11c⁺ fractions correspond toconventional dendritic cells (cDCs), which are further divided in CD11b⁻cDCs, either migratory (CD103⁺, gate 4) or resident (CD103⁻, gate 5,FIG. 6G); and CD11b⁺ cDCs, again either migratory (CD103⁺, gate 7) orresident (CD103⁻, gate 8, FIG. 6H). An additional population ofCD103⁻SIRPα⁺ CD11b⁻ cDCs not detailed in the literature was alsovisualized (gate 6, FIG. 6G). When comparing PPI-Fc- and IgG1-fed2-week-old mice, no differences were highlighted in the frequency orcounts of these different APC populations (not shown). Collectively,these results show that APC composition is not influenced by prior oralvaccination, at least at the 2-week time point analyzed.

The T-cell gating strategy is depicted in FIG. 7. Among lineage⁻CD3⁺cells, CD8⁺ T cells were gated (FIG. 7A, gate 1). Among CD8⁺ cells, MMr⁺cells were selected to verify their specificity for the PPI_(B15-23)peptide (FIG. 7B). CD8⁺ T cells were further gated for CD44 and CD62L.While the CD44^(hi)CD62L⁻ subset corresponds to activated/memory CD8⁺ Tcells (gate 2), CD44⁻CD62L⁺ are naïve cells (gate 3, FIG. 7C). Finally,both activated and naïve CD8⁺ T cells were analysed for the expressionof the gut-homing markers CCR9 and α4β7 (gates 4-5, FIG. 7D; and gates6-7, FIG. 7E). For CD4⁺ T cells (FIG. 7F), the same phenotypic markers(CD44 and CD62L, FIG. 7G; and CCR9 and α4β7, FIG. 7H-I) were used. Inaddition, three Treg populations were analysed: classical Tregs, eitherthymic-derived (Foxp3⁺NPR1⁺; gate 15) or peripherally induced(Foxp3⁺NRP1⁻; gate 16, FIG. 7J); T helper (Th)3 cells (LAP⁺FoxP3⁻; gate17, FIG. 7K) and T regulatory 1 (Tr1) cells (Foxp3⁻CD49b⁺LAG3⁺; gate 18,FIG. 7L). Using this gating strategy, few differences between PPI-Fc-and IgG1-fed animals were already visible at 2 weeks of age. First, theproportion of splenic CCR9^(hi)CD8⁺ T cells, which are likely tooriginate in the gut, was increased in PPI-Fc-fed mice (FIG. 8). Suchincrease was confined to activated CCR9^(hi)CD8⁺ T cells (FIG. 8A-B) andwas not observed in the naïve CD8⁺ subset (FIG. 8C-D), furthersuggesting that T cells migrate to the spleen from the gut uponencounter with their PPI B15-23 cognate antigen that is recognized bymost T cells in this G9C8 TCR -transgenic model. Second, a similarincrease in the PPI-Fc group was observed for splenic α4β7±CD4⁺ T cells(FIG. 9), another population reported to originate in the gut. Also inthis case, this increase was observed only in the activated (FIG. 9A-B)but not in the naïve subset of these cells (FIG. 9C-D), likelyreflecting prior cognate PPI-Fc priming in the gut. Third, a minorincrease in splenic peripheral Tregs was observed with the PPI-Fctreatment (FIG. 10A-B), while splenic thymus-derived Tregs were notincreased at this time point (FIG. 10C-D).

Collectively, these results show that oral PPI-Fc vaccination inducesT-cell modifications characteristic of oral tolerance, namely anincrease in gut-derived activated CD8⁺ and CD4⁺ T cells and inperipheral Tregs at 2 weeks of age; and modifications suggestive ofdeletional and regulatory tolerance mechanisms, namely decreased CD8⁺and CD4⁺ effector T cells and increased CD4⁺ Tregs at 4 weeks. Offurther note, the proposed mechanism of action for oral PPI-Fcvaccination is different than for classical oral tolerance withFc-devoid antigens. Systemic and thymic antigen-Fc bioavailability ishere achieved, boosting both peripheral and central tolerancemechanisms, as evidenced by the increased numbers of both thymic- andperipheral-derived Tregs.

Neonatal Oral PPI-Fc Vaccination Protects G9C8 Mice from DiabetesDevelopment

Finally, we verified whether PPI-Fc oral vaccination and the associatedT-cell modifications resulted in diabetes protection later in life. Tothis end, 1-day-old newborn G9C8 mice were orally vaccinated with 50 μgPPI-Fc. At 4 and 6 weeks of age, they were then immunized with PPIB15-23 peptide and CpG to induce diabetes and prospectively followed.For controls, equimolar amounts of recombinant IgG1 (i.e., irrelevantprotein with preserved FcRn binding) and PPI (i.e., cognate antigen withno FcRn binding) were administered. In IgG1-fed mice, diabetesdevelopment was rapid and synchronous with prime-boost immunizations,affecting 93% of mice. Conversely, only 44% of mice developed diabeteswhen fed with PPI-Fc (p<0.01). As expected, PPI gave an intermediateprotection, with 72% of mice ultimately developing diabetes.Collectively, these results demonstrate that oral vaccination withPPI-Fc protects G9C8 mice from diabetes more efficiently than Fc-devoidPPI.

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1. A method for inducing tolerance to one antigen of interest in asubject in need thereof, comprising the mucosal administration to thesubject of a therapeutically effective amount of a recombinant chimericconstruct comprising a FcRn targeting moiety and an antigen-containingmoiety.
 2. The method of claim 1 wherein the antigen is an auto-antigen,the antigen is an allergen or the antigen is a molecule that isexogenously administered for therapeutic purposes.
 3. The method ofclaim 1 wherein the subject is an adult, a pregnant woman or a child. 4.The method of claim 1 wherein the subject is a newborn or a neonate. 5.The method of claim 1 wherein the subject is predisposed or believed tobe predisposed to developing, or has already developed or is developingan autoimmune disease.
 6. The method of claim 1 wherein the subject ispredisposed or believed to be predisposed to developing, or has alreadydeveloped or is developing an allergy.
 7. The method of claim 1 whereinthe subject is predisposed or believed to be predisposed to developing,or has already developed or is developing an immune reaction againstmolecules that are exogenously administered for therapeutic or otherpurposes.
 8. The method of claim 1 wherein the subject is predisposed orbelieved to be predisposed to developing, or has already developed or isdeveloping an immune reaction against a grafted tissue or graftedhematopoietic cells or grafted blood cells.
 9. The method of claim 1wherein the FcRn targeting moiety is an Fc of an IgG antibody,preferably of an IgG1 or IgG4 antibody, even more preferably of an IgG1antibody, or a portion of the Fc.
 10. The method of claim 1 wherein therecombinant chimeric construct of the present invention is a fusionprotein that comprises an amino acid sequence consisting of a portion ofan Fc region and an amino acid sequence that comprises the antigenicportion of the antigen.
 11. The method of claim 1 wherein therecombinant chimeric construct is administered to the subject with themeans of recombinant bacteria that express the construct.
 12. The methodof claim 1 wherein the recombinant chimeric construct is delivered viathe oral cavity.
 13. The method of claim 1 wherein the recombinantchimeric construct is delivered via the respiratory tract.
 14. Themethod of claim 13, wherein the recombinant chimeric construct isdelivered via the nasal cavity.